Effects of TmTak1 silencing on AMP production as an Imd pathway component in Tenebrio molitor

Mealworms beetles, Tenebrio molitor, are the limelight next-generation food for humans due to their high nutrient contents. Since Tenebrio molitor is used as feed for pets and livestock in addition to their ability to decompose polystyrene and plastic waste, it is recognized as an insect with an industrial core value. Therefore, it is important to study the immune mechanism related to the development and infection of mealworms for mass breeding purposes. The immune deficiency (Imd) signaling is one of the main pathways with pivotal roles in the production of antimicrobial peptides (AMPs). Transforming growth factor-β activated kinase (TAK1) is one of the Imd pathway components, forms a complex with TAK1 binding protein 2 (TAB2) to ultimately help activate the transcription factor Relish and eventually induce host to produce AMPs. Relatively, little has been revealed about TAK1 in insect models, especially in the T. molitor. Therefore, this study was conducted to elucidate the function of TmTak1 in T. molitor. Our results showed that the highest and lowest mRNA expression of TmTak1 were found in egg and young larvae respectively. The tissue-specific expression patterns were reported in the gut of T. molitor larvae and the fat bodies of adults. Systemic microbial challenge illustrated TmTak1 high expression following the fungal infection in all dissected tissues except for the whole body. However, silencing TmTak1 experiments showed that the survivability of T. molitor larvae affected significantly following Escherichia coli infection. Accordingly, AMP induction after TmTak1 knock down was mainly reported in the integument and the fat bodies.

The yellow mealworm, Tenebrio molitor (T.molitor) is a well-known insect not only because of its high nutritional values but also as a crop pest 1,2 .T. molitor exposure to different pathogenic factors in their natural inhabitants has made them a great immune study model [3][4][5] .Considering T. molitor industrial importance, their relatively larger size, and their convenient rearing condition, it is important to discuss disease resistance defense mechanisms related to the development and health of mealworms 6,7 .Like other insects, yellow mealworms have unique physical and physiological mechanisms to prevent foreign invaders or reduce infection 3 .Among all the shields, T. molitor uses its innate immune arms to produces antimicrobial peptides (AMPs) through a specific mechanism [8][9][10][11][12] .Insects' innate immunity perceive Lipopolysaccharide (LPS) and Peptidoglycan (PGN) from various microbial sources via their pattern recognition receptors (PRRs) 13,14 .Functional immune response is divided into cellular and humoral immunity 15 .Cellular immunity includes blood cell-mediated immune responses including phagocytosis, nodulation, and encapsulation 16 , whereas humoral immunity involves Toll, immune deficiency (Imd), and the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways triggering immune related gene expression including but not limited to AMPs production 5,[17][18][19][20][21] .While in Drosophila both Toll and Imd pathways are important, Imd pathway regulates most of the AMPs expression 18 .In Drosophila, peptidoglycan recognition proteins (PGRPs) which recognize bacterial cell wall compartments can be divided into PGRP-SA, PGRP-LC, and PGRP-LE 22 .Lys-type PGN, a cell wall component of Gram-positive bacteria, is recognized by PGRP-SA 23 , activates the Toll pathway, and induce AMPs production.On the other hand, DAP-type PGN, a cell wall component of Gram-negative bacteria, is recognized by PGRP-LC and LE 24

In silico analysis of TmTAK1
As a result of homology mapping of the TmTak1 cDNA sequence based on the T. molitor DNA-seq database, 8 exons intervening with 7 introns were identified (Supplementary Fig. 1).Among the 8 exons of TmTak1, exon 2 was the longest with 330 sequences, followed by exon 4 with 249 nucleotide sequences and exon 8 with 220 nucleotide sequences.Among the 7 introns, intron 6 was longer than the other introns, so the size of the introns was not uniform.In addition, the coding sequence consisted of nucleotides of 1527 bp and coded 508 amino acid residues.The full-length coding sequence of TmTak1 was submitted to GenBank, accession number OR373077 has been granted.As a result of protein domain analysis of TmTAK1, it was confirmed that the protein tyrosine kinase domain from amino acid residues no.24 to 256 and four tyrosine kinase catalytic domains were located at no. 93 to 106, no.134 to 152, no.179 to 189, and no.247 to 269 residues (Supplementary Fig. 2).TAK1 orthologues and Multiple Sequence Alignment (MSA) of insect was analyzed using ClustalX v2.0.Through the MSA results, it was confirmed that protein tyrosine kinase and tyrosine kinase catalytic domains in the TmTAK1 amino acid sequence is well conserved in other insects (Supplementary Fig. 3).The phylogenetics of TmTAK1 were analyzed using TAK1 homologues from other insect species (Supplementary Fig. 4).TmTAK1 clustered with TcMKKK7 which has 85% sequence identity with TmTAK1 and was isolated under the same cluster as other coleopteran species.

Developmental and tissue-specific expression patterns of TmTak1
During the development of T. molitor, the expression pattern of TmTak1 was investigated by qPCR.As a result, the expression level of TmTak1 was highest in young larvae (YL) and lowest in 7-day old pupae (P7) (Fig. 1A).As a result of examining the tissue-specific expression pattern of TmTak1 in T. molitor, high expression level of TmTak1 was confirmed in the gut (GT) of the larval stage and low expression level was confirmed in the integument (INT) (Fig. 1B).Next, in the adult stage, the expression level of TmTak1 was the highest in the fat bodies (FBs), and low expression levels were confirmed in the other tissues (Fig. 1C).

TmTak1 mRNA expression level after microbial challenge
T. molitor larvae were infected with PBS, E. coli, S. aureus, and C. albicans, and after 3 h, 6 h, 9 h, 12 h, and 24 h, WB and five tissues (GT, FBs, MTs, INT, and HCs) were dissected (Fig. 2).TmL27a was used as a house keeping gene, and the relative expression level of TmTak1 was investigated using qPCR using PBS as a control.As a result of examining the expression level, high expression levels specifically for C. albicans were confirmed in the gut, fat bodies, Malpighian tubules, integument, and hemocytes compared to E. coli in whole body.In particular, the expression level of TmTak1 was relatively higher in MTs than other tissues against C. albicans infection (Fig. 2E).

TmTak1 knockdown increased the mortality of E. coli-injected T. molitor larvae
The mortality was investigated after RNAi using dsTmTak1 in T. molitor (Fig. 3A).The concentration of bacteria used in the experiment was 1 × 10 7 CFU/μl for E. coli and S. aureus, and 5 × 10 5 CFU/μl l for C. albicans, and n = 10 were repeated three times.It was confirmed up to the 10th day after infection with the microorganisms (Fig. 3B-D).There was no significant difference between S. aureus and C. albicans, but after E. coli infection, the survival rate of the group injected with dsTmTak1, the experimental group, was reduced to about 10% on the first day and 35% on the second day, compared to the control group injected with dsEGFP (Fig. 3B).

Effects of TmTak1 RNAi on the expression of Tenebrio NF-κB genes
The expression of T. molitor NF-κB genes implicated in the IMD (TmRelish), JNK (TmKayak), and Toll (TmDorX1 and TmDorX2) pathways were studied in TmTak1-silenced individuals upon microbial challenge (Fig. 8).The study was conducted to address the antimicrobial activity of TmTak1.The mRNA expression of TmRel and TmKay transcripts were significantly decreased in the MTs, GT, and HCs of TmTak1-silenced T. molitor in a descending manner.Conversely, relevant expression of TmRel upregulated in the TmTak1 silenced INT and FBs, suggesting involvement of TmTak1 in the canonical pathway of AMP production which effects TmRel mRNA expression MTs, GT, and HCs.Furthermore, the relevant downregulation of TmRel and TmKay mRNA expression was more significant after E. coli challenge compared to S. aureus and C. albicans challenge.Regarding the TmDorX1 and TmDorX2 identical result was reported, while the most significant depletion of transcription factor expression was related to TmDorX2 following E. coli challenge in the GT of silenced larvae.Accordingly, these results justified the significant mortality of the T. molitor larvae following E. coli challenge.

Discussion
The innate immune system is the first line of defense against invading pathogens and is comprised of a variety of cellular and humoral components 35 .In terms of immunological effects, TAK1 has been shown to play a critical role in the regulation of the innate immune response in insects 20,25,[36][37][38] .In this study, we found that TAK1 might be as regulator Imd and JNK signaling pathway which response to pathogenic infection such as E. coli.TAK1 is a serine/threonine kinase that is involved in a wide range of signaling pathways in various organisms, including mammalian and insects [39][40][41] .In mammalian, TAK1 is complexed with adaptor proteins TAB1, TAB2 and TAB3.TAB1 interact with TAK1 and triggers autophosphorylation of TAK1, which is essential for TAK1 kinase activity.TAB2 and TAB3 are involved in complex structure and share 48% of both amino acid sequence.TAB1 binds to the kinase domain in N-terminus of TAK1, and TAB2 and TAB3 bind to the C-terminal region of TAK1 42 .Complex between the kinase domain of TAK1 and the C-terminal TAB1 stimulates phosphorylationdependent TAK1 activation 43 .Herein, we found TmTAK1 includes the protein tyrosine kinase domain from amino acid residues no.24 to 256 and four tyrosine kinase catalytic domains were located at no. 93 to 106, no.134 to 152, no.179 to 189, and no.247 to 269 residues (Supplementary Fig. 2).Further, the sequence alignment results revealed the presence of homologs of TAK1 in many insect species, this study assigned Odonata, Isoptera, Hemiptera, Hymenoptera, Coleoptera, Lepidoptera, and Diptera (Supplementary Fig. 3) and phylogenetic characteristics of TmTAK1 disclose adjacent association within the Coleoptera (Supplementary Fig. 4).
In this study, mRNA of TmTak1 was expressed in all developmental stage and the highest level of expression was detected in Egg and YL stages (Fig. 1A).In Xenopus embryos, xTAK1 and xTAB1 is relative with ventral mesoderm development.The ventralization caused by constitutively active the bone morphogenetic protein receptor IA (BMPR-IA) and Smad1/5 is reversed by the kinase-negative form of xTAK1.Ectopic expression     www.nature.com/scientificreports/ of xTak1 led cell death in early embryos 44 .It must be borne in mind that previous studies in Drosophila and silkworm demonstrated that during the developmental stages and metamorphosis further to pathogenic infections 20-hydroxyecdysone (20E), one of the two most important developmental hormones, improves the insect innate immune response, via upregulation of the PGRP-LCs 45,46 .Accordingly, our findings also support the solid interactions of developmental and immune system compartments.
TmTak1 is required for activation of both Imd and JNK NF-κB after three type of pathogen such as Gram negative-, Gram positive-bacteria, and Fungi infection (Fig. 2).In Drosophila, PGN stimulation triggered TAK1 activation in both NF-κB and JNK mitogen-activated protein kinase (MAPK) signaling 25,37,47,48 .In Drosophila infected with Gram-negative bacteria, negative regulation of JNK signaling was demonstrated to be associated with proteasomal degradation of TAK1 36 .In crustacean, TAK1 activates the Imd and JNK pathways in response to Gram negative infections 49 .The endogenous TAK1 is phosphorylated in LPS-stimulated RAW 264.7 cells 50 .In mammalian, TAK1 is critical mediator of the antifungal immune response and a key component of the signaling cascade downstream of the c-type lectin receptor 51 .
The pathogen-derived metabolite, phenazine-1-carboxamide, has recently been discovered to be recognized by nuclear hormone receptors and to induce anti-pathogen defense in C. elegans 52 .Because a live microorganism was employed in this study, it is possible that the bacterial metabolite activates a non-canonical immunological pathway.Moreover, it has be reported repeatedly that various bacterial metabolite compositions which differ in distinct bacterial species including but not limited to E. coli might sequentially and cooperatively interfere with humoral and cellular defense response [53][54][55] .While these secondary metabolites might highly effect immune related signaling pathways via induction of host immunosuppression little is known about the relevant interaction.Hence, more research is needed to clarify the effect of bacterial metabolites on the immune system of T. molitor.
In T. molitor, TAK1 has been shown to be involved in the regulation of immune-related processes.For example, TAK1 activation has been shown to induce the expression of AMPs in T. molitor larvae.After infection with bacterial and fungal pathogens, the highest expression levels of TmTak1 were observed specifically in response to C. albicans infection in multiple tissues including the gut, fat bodies, Malpighian tubules, integument, and hemocytes (Fig. 2).In mosquito, two entomopathogenic fungi Beauveria bassiana and Isaria javanica trigger Imd pathway component and induce AMPs in the gut and fat bodies 56 .In mice, fungal infection induce JNK1 which negatively controls innate immune response by suppressing CD23 expression 57 .
Like Drosophila, AMP production is regulated by Dorsal and Relish, which are transcription factors of Toll and Imd signaling pathways [58][59][60] .Moreover, dTAK1 is a factor involved in the JNK pathway 36 .JNK pathway is one of the main conserved signaling branch of the MAPK signaling pathway 61,62 .Kayak is one of the components of AP-1 transcription factor complex 63 .The sequences of TmKayak were retrieved from the T. molitor RNAseq and expressed sequence tag (EST) databases using local-tblastn analysis (unpublished).After gene silencing of TmTak1, the mRNA expression pattern of AMPs was significantly decreased by pathogen infection, proving that TmTAK1 plays a critical role in AMP production.In order to confirm which transcription factors caused the absence of AMP expression due to gene silencing of TmTak1, the expression levels of Toll, Imd, and JNK former factors were examined.Despite systemic infection with E. coli and S. aureus, TmRel and TmKay were significantly decreased after TmTak1 silencing in the MTs proposing critical roles of TmRel and TmKay in AMP induction.According to our finding, Gram-negative bacteria infection in shrimp revealed that TAK1 activated AMP production via c-jun and Relish 49 .
Knockdown of TmTak1 using RNA interference resulted in increased mortality in T. molitor larvae following E. coli infection (Fig. 3B).TAK1 activates downstream kinase the inhibitor of NF-κB (IκB) kinase (IKK) complex 64 .Several studies have been conducted on immunological roles of IKK complex components [65][66][67] .For instance it has been shown that similar to TmTak1, knock-down of TmIKKε causes higher mortality rate post E. coli systemic infection 67 .Moreover, TmIKKβ and TmIKKγ/NEMO are critical immune factor for T. molitor survival following E. coli, S. aureus and C. albicans infections 65,66 .
Our observations bring out the importance of reaching a better perspective over TmTAK1 regulatory acts on each IKK components independently and IKKs complex.In addition, the direct action of TmTAK1 on components of the JNK pathway remains to be elucidated.This study suggests that TmTAK1 plays a role in the innate immune response of T. molitor to Gram negative infection and also triggers Imd and JNK pathway.

Insect rearing
Insects were reared in a plastic container measuring 29 cm in width, 21 cm in length, and 9.5 cm in height, and wheat bran was used as food.The mealworms used in the experiment were separately fed to an artificial diet (170 g wheat flour, 20 g roasted soy flour, 10 g protein, 100 g wheat bran, 0.5 g sorbic acid, 0.5 mL propionic acid, and 0.5 g chloramphenicol in 200 mL of distilled water) and sterilized water was supplied once a day to maintain the temperature of 26 °C ± 1 °C and humidity of 60% ± 5% in an incubator.The larvae used in all experiments except for the developmental experiment were 10-12 years old (1.34-1.88cm), and the adult was 5 days old after eclosion.

Identification and in silico characterization
The sequences of TmTak1 were retrieved from the T. molitor RNAseq (unpublished) and expressed sequence tag (EST) databases using local-tblastn analysis.For the retrieval, the amino acid sequences of Tribolium castanuem TAK1 (TcTAK1) (XP_968547. 1) were used as a query.For TmTak1 gene architecture, local-tblastn analysis was performed with TmTak1 nucleotide sequence as query against the T. molitor DNASeq database (unpublished) as the subject.Conserved domains were identified using InterProScan (https:// www.ebi.ac.uk/ inter pro/

Figure 2 .
Figure 2. TmTak1 mRNA expression profiles after microbial challenge.Expression of TmTak1 mRNA in the whole body (A), hemocytes (B), gut (C), fat bodies (D), Malpighian tubules (E), integument (F) of larvae infected with Escherichia coli, Staphylococcus aureus, and Candida albicans.The expression was analyzed by qPCR using L27a (T.molitor) as the internal control.For each time point, the expression level in the phosphatebuffered saline (PBS)-injected control (mock control) was set to 1; this is represented by a dotted line.vertical bars represent mean ± standard error from three biological replicates.

Figure 3 .
Figure 3.Effect of TmTak1 RNAi on T. molitor Larval Survival.(A) TmTAK1 knockdown efficiency measured using RT-qPCR at day 5 post-injection.The survivability of larva was measured after TmTak1 knockdown and infection with Escherichia coli (B), Staphylococcus aureus (C), or Candida albicans (D) (n = 30).Data are presented as average of three biologically independent replicate experiments.Asterisks indicate significant differences between TmTak1-knocked down and dsEGFP-treated groups (p < 0.05).Survival rates were analyzed based on the Kaplan-Meier plots (log-rank chi-square test; *p < 0.05).